Although a relatively new area of study, micromachined chemical analysis systems have the potential to revolutionize separation methods, much like integrated circuit manufacturing transformed advanced computation in the twentieth century. Miniaturized devices promise to reduce both reagent consumption and cost while simultaneously increasing separation speed and throughput. Already, microdevices have been utilized in clinical diagnostics41, analytical chemistry42, human genome research43, and in the pharmaceutical industry,44 allowing new access to information on the molecular level.
Miniaturization in chemical separations had its start in the late 1970's when Terry et al.45 microfabricated an integrated gas chromatography system on a silicon wafer. While this initial work went largely unnoticed for more than a decade, efforts in this area were renewed in the 1990's with the publication of a conceptual miniaturized total chemical analysis system in 199046 and the development of planar microfabricated capillary electrophoresis (CE) substrates in 1992.47 These studies sparked an increasing interest in miniaturization, creating a demand for devices with ever-improving functionality at ever-lower costs.
The use of polymeric microchip substrates has been an important advance. Currently, glass devices predominate for a number of reasons. First, the chemistry of glass surfaces is well understood. Second, glass is transparent to visible light wavelengths and has low intrinsic fluorescence,48 a critical characteristic, since detection often involves monitoring of optical signals, such as fluorescence, UV absorbance, or Raman scattering. Finally, photolithographic processes already used for silicon materials can be readily adapted to glass substrates. Despite these advantages, there are also several significant drawbacks to working with glass. For example, substrate costs for glass can exceed those for common polymers by a factor of 10 to 100 (see Table 1). Expenses also accumulate during the many steps involved in preparing glass substrates, such as cleaning, photoresist coating, photolithography, development, etching, etc., making the fabrication process not only pricey, but also complicated and time consuming. Furthermore, microcapillary formation in glass materials requires a high-temperature (>600° C.) annealing step. Finally, once a substrate is fully prepared, a surface modification procedure is often necessary because many biomolecules can adsorb to native glass.
TABLE 1Prices for polymers and different types of glass.49, 50MaterialCents per cm2Poly(methyl methacrylate) (PMMA)0.2-2 Polycarbonate (PC)0.5-4 Borofloat glass10-20Borosilicate glass 5-15Photostructurable glass20-40
Research into replacing glass in microdevices with polymers began in the mid-1990's. An all-plastic microfluidic system was reported in 1996 by VerLee et al.,51 however, the channel dimensions were still in the range of conventional fused silica capillary diameters (˜100 μm). Shortly thereafter researchers began to develop smaller (15-20 μm) plastic microchannels.52 Because plastics are typically less expensive, more biocompatible, and easier to manipulate than silica- or silicon-based materials, the development of suitable polymer microfabrication methods continues to be important for the production of low-cost, disposable miniaturized devices.
As the lab-on-a-chip field has developed, certain polymers such as poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA) and polycarbonate (PC) have been employed increasingly as device substrates.1-3 This shift toward polymeric substrates has likely occurred because of two factors. First, the templated procedures used to create microchips in polymers allow a single photolithographically defined master to be used to pattern numerous devices,5,6 thus decreasing the need for cleanrooms and other costly instrumentation. Second, the polymeric materials themselves are typically less expensive than microchip-quality glass, and lower costs per device should facilitate the development of disposable microfluidic systems.3 
Despite these attractive features of polymeric materials, glass remains the substrate of choice for very fast7 or high-performance8,9 microchip CE. This performance gap is due in part to the convenience of adapting the well-characterized chemistry of fused silica capillaries for surface modification in a wide array of glass microchip applications. Also, the thermal conductivity of glass is higher than that of commonly used polymers (e.g. PMMA, PC and PDMS),10 which provides better dissipation of Joule heating and enables higher electric fields in microchannels in glass substrates.
Unfortunately, glass microchips must be patterned and etched individually in a cleanroom, and the thermal annealing of glass substrates to enclose microcapillaries generally takes place in a furnace at >400° C. for several hours.1 Moreover, special care must be taken to ensure that the bonded surfaces are extremely clean and lacking even small particulates, or thermal bonding will not be successful.11 Low- and room-temperature glass bonding approaches that avoid high-temperature processing have been reported,12-15 but the resulting adhesion is weaker than in thermally sealed devices, and even greater care must be taken to ensure that the surfaces are extremely clean and flat.
To avoid a sealing step for microcapillary enclosure and to create sub-μm features, sacrificial techniques have been explored. In these methods a channel design is patterned on top of a bulk substrate, and a thin film of a different material is deposited over the entire surface, covering the patterned design. Next, the sacrificial material under the deposited layer is etched away16,17 or thermally decomposed,18-21 leaving microcapillaries defined by the cover layer and the base substrate. While these sacrificial methods have successfully created devices without a thermal bonding step, the fabrication protocols are involved, and templated procedures are not possible because each device is patterned individually.
Other approaches for microfluidic device construction involve the creation of capillaries by affixing a cover plate to a surface containing microfabricated channels. Several approaches for sealing polymer substrates have been utilized to form microcapillaries, including heating in a convection oven,52,53 placement under heated weights54 or in boiling water,55 thermal lamination,56 and adhesion with tape57 or poly(dimethylsiloxane) (PDMS) films.58 While adhesive tapes and elastomer films are convenient, thermal-bonding approaches continue to predominate because they enable the formation of microcapillaries with a uniform surface composed entirely of the same polymeric material. Since substances have characteristic surface charge distributions (or zeta potential), when a channel is comprised of different top and bottom materials, as is necessitated by adhesive and elastomer bonding, the different surface properties result in unwanted band broadening in separations. On the other hand, the temperatures necessary for thermal bonding, normally >100° C. for polymers, often result in undesired reactions of microchip surface coatings. Because of these limitations, the development of new bonding techniques remains an important focus of microchip fabrication research.
Phase-changing materials (typically waxes) have been incorporated into fluidic microchips to create micropumps,22 membrane actuators23 and valves,24,25 but these materials have not been used as sacrificial layers in microdevice fabrication. In addition, Liu, et al.24 recently used a solvent-assisted thermal bonding method to seal PC substrates at ˜200° C., but the large feature dimensions (>300 μm deep) made it unnecessary to protect the channels from the bonding solvent.
Microfluidic devices have made possible extremely fast72 and high-performance73-75 chemical separations, but perhaps the most significant promise of lab-on-a-chip technology is in the ability to combine multiple sample handling and analysis steps onto a single miniaturized platform.76-79 Such integration can decrease the total analysis time significantly, especially with complex samples that require extensive pretreatment. For example, microfluidic mixers and reactors have been combined on microchip capillary electrophoresis (μ-CE) devices having six parallel separation lanes to perform multiple immunoassays in ˜1 min.80 In another case μ-CE systems with polymerase chain reaction chambers having on-chip heaters, temperature sensors and valves facilitated genotyping from whole bacterial cells in <10 min.81 However, one challenge associated with μ-CE is concentration sensitivity, since small sample volumes are loaded in these systems typically. Thus, it can be advantageous to integrate sample clean up and preconcentration on-chip to enhance the signal intensity from dilute specimens.
As size-selective membranes can facilitate many sample preparation and manipulation steps, researchers have focused on interfacing membranes with microfluidics. For example, Smith and coworkers82 created a microdialysis system that sandwiched commercially available sheet membranes between microchannel-containing substrates, allowing samples to be purified from interfering high- and low-molecular weight species prior to being introduced into a mass spectrometer. In a similar setup affinity microdialysis was performed on-chip; antigen-antibody complexes were retained by a sheet membrane while smaller, unbound components were removed.83 Then, the purified complexes were exposed to counterflowing air through a second membrane, which concentrated the sample through solution evaporation. Nanoporous track-etched polycarbonate membranes were utilized in interfacing microchannels on different substrates.84-86 Analyte transport between channels was controlled by applying an electric field across the membrane, enabling selected fractions from one channel network to be driven electrokinetically through the nanopores and introduced into the opposing channel structure. Samples were injected and fractions were collected across a membrane using this approach, showing considerable control in analyte manipulation. Ramsey et al.87,88 demonstrated size-selective barriers for DNA concentration prior to electrophoretic separation. Channels in a μ-CE injector were connected electrically through small pores in a thin sodium silicate layer. DNA molecules that were driven electrokinetically to the sodium silicate membrane were too large to pass through, and over time the concentration of the trapped DNA increased ˜100-fold; the enriched sample plugs were then separated electrophoretically. Zhang and Timperman89 employed a conceptually similar preconcentration system that had a sandwiched, track-etched membrane. Rather than pore size, charge played the dominant role in analyte trapping, as the 10-50 nm through holes were much larger than the molecules that were enriched. While these examples demonstrate the broad applicability of membrane-based microsystems to various modes of sample pretreatment and manipulation, most utilized commercial sheet membranes sandwiched between microfluidic device substrates. Such configurations have limited device geometries and are constrained by the properties of available materials.
The ability to polymerize semi-permeable barriers in situ in microfluidic networks adds design flexibility and enables membranes with a variety of properties to be explored. Recently, a dialysis system that incorporated an in situ-polymerized membrane was reported by Kirby and coworkers.90 A microchannel was filled with a prepolymer solution having an appropriate photoinitiator, and a laser beam was focused into a plane to effect spatially controlled polymerization. This produced a membrane that divided the channel in two along its length, allowing dialysis to take place between countercurrent flows. Membrane properties could be altered by tailoring the prepolymer composition, but a complicated optical setup was required, and repeated laser exposures with fresh monomer solution in the channels were necessary to complete polymerization. Thus, improved methods are still needed for the convenient creation of polymer membranes in microfluidic networks.
Electric field gradient focusing (EFGF) is an analytical technique that is facilitated by having a semi-permeable membrane interfaced with a separation column.91-97 Briefly, a gradient in electric field, combined with a constant-velocity pressure-driven flow in the opposite direction, causes charged analytes to focus into stationary bands along the column according to electrophoretic mobility. A capillary-based EFGF design that interfaced an in situ-polymerized semi-permeable copolymer (SPC) of changing cross-sectional area (CSA) with a ˜100 μm-diameter focusing column has been reported.95 The SPC permitted current-carrying buffer ions to pass through, but the bulk fluid and protein analytes could not. The focusing column was formed by polymerizing the SPC around a wire in a well of changing CSA. After polymerization the wire was pulled out from one of the ends of the microchip, leaving an open cylindrical column connected to capillaries at both sides of the SPC. Although this approach allowed for smaller-dimension devices than previous membrane-incorporating EFGF designs,91-93,97 several limitations were also apparent. For example, further column miniaturization was impractical, as thinner wires were more fragile and difficult to use. In addition, a diameter mismatch between the focusing channel and the capillaries would reduce resolution if analytes were eluted from the column. Improved EFGF device fabrication methods that avoid these challenges, while enabling smaller channel dimensions, would be valuable.
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